Se-Jin Lee

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β–Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload–induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload–induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast–specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β–Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit muscle mass. In adult animals, MSTN is made almost exclusively by skeletal muscle and circulates in the blood. A critical question is whether this circulating MSTN protein can enter the active pool to regulate muscle growth or whether all of the activity of MSTN results from locally produced protein. Here, we addressed this question in mice by using a Cdx2-Cre transgene in conjunction with a conditional Mstn-flox allele to generate mice in which Mstn was targeted in a regionally restricted manner. Specifically, we generated mosaic mice in which MSTN production was eliminated in posteriorly located muscles but not in anteriorly located muscles, resulting in mice in which circulating levels of MSTN were reduced roughly by half. Analysis of posteriorly located vs. anteriorly located muscles of these mice revealed clear differential effects indicative of an important paracrine role for MSTN in regulating muscle mass. Significant, albeit more subtle, effects consistent with an endocrine mode of MSTN action were also seen in these mice. These findings have important implications not only for the understanding of the physiological control of muscle mass but also for therapeutic strategies to target MSTN to treat patients with muscle loss.

Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf^−/−) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf^−/− mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf^−/− mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf^−/− mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf^−/− mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis.

Myostatin (MSTN) and growth and differentiation factor-11 (GDF-11) are highly related TGF-β family members that have distinct biological functions. MSTN is expressed primarily in skeletal muscle and acts to limit muscle growth. GDF-11 is expressed more widely and plays multiple roles, including regulating axial skeletal patterning during development. Several MSTN and GDF-11 binding proteins have been identified, including GDF-associated serum protein-1 (GASP-1) and GASP-2, which are capable of inhibiting the activities of these ligands. Here, we show that GASP-1 and GASP-2 act by blocking the initial signaling event (namely, the binding of the ligand to the type II receptor). Moreover, we show that mice lacking Gasp1 and Gasp2 have phenotypes consistent with overactivity of MSTN and GDF-11. Specifically, we show that Gasp2^−/− mice have posteriorly directed transformations of the axial skeleton, which contrast with the anteriorly directed transformations seen in Gdf11^−/− mice. We also show that both Gasp1^−/− and Gasp2^−/− mice have reductions in muscle weights, a shift in fiber type from fast glycolytic type IIb fibers to fast oxidative type IIa fibers, and impaired muscle regeneration ability, which are the reverse of what are seen in Mstn^−/− mice. All of these findings suggest that both GASP-1 and GASP-2 are important modulators of GDF-11 and MSTN activity in vivo. Significance The TGF-β family encompasses a large number of secreted proteins that regulate embryonic development and adult tissue homeostasis. Growth and differentiation factor (GDF) -associated serum protein-1 (GASP-1) and GASP-2 are related proteins capable of binding and inhibiting two family members, myostatin and GDF-11. Here, we show that mice genetically engineered to lack GASP-1 and/or GASP-2 exhibit muscle and skeletal phenotypes consistent with overactivity of myostatin and/or GDF-11. These studies also reveal the enormous complexity of this regulatory system in vivo and the delicate balance that must be maintained between signaling molecules and their inhibitory proteins for proper levels of signaling to be achieved.

Myostatin and activin A are structurally related secreted proteins that act to limit skeletal muscle growth. The cellular targets for myostatin and activin A in muscle and the role of satellite cells in mediating muscle hypertrophy induced by inhibition of this signaling pathway have not been fully elucidated. Here we show that myostatin/activin A inhibition can cause muscle hypertrophy in mice lacking either syndecan4 or Pax7, both of which are important for satellite cell function and development. Moreover, we show that muscle hypertrophy after pharmacological blockade of this pathway occurs without significant satellite cell proliferation and fusion to myofibers and without an increase in the number of myonuclei per myofiber. Finally, we show that genetic ablation of Acvr2b , which encodes a high-affinity receptor for myostatin and activin A specifically in myofibers is sufficient to induce muscle hypertrophy. All of these findings are consistent with satellite cells playing little or no role in myostatin/activin A signaling in vivo and render support that inhibition of this signaling pathway can be an effective therapeutic approach for increasing muscle growth even in disease settings characterized by satellite cell dysfunction.

Skeletal muscle wasting is a major component of cachectic states found in a variety of disease settings, including cancer. As increasing caloric intake often provides little benefit in combating muscle loss in cachectic patients, a major research focus has been to develop strategies stimulating muscle anabolic pathways - in an attempt to fight the catabolic pathways induced during cachexia. Two recent papers have reported the beneficial effects of blocking the myostatin/activin signalling pathway in mouse models of cancer cachexia. We discuss the implications of their findings both with respect to the role that this signalling pathway may play in the aetiology of cachexia and with respect to the prospects for targeting this pathway as a therapeutic strategy in patients with cachexia.

Myostatin (MSTN) is a transforming growth factor-β family member that plays a critical role in regulating skeletal muscle mass. Genetic studies in multiple species have demonstrated that mutations in the Mstn gene lead to dramatic and widespread increases in muscle mass as a result of a combination of increased fiber numbers and increased fiber sizes. MSTN inhibitors have also been shown to cause significant increases in muscle growth when administered to adult mice. As a result, there has been an extensive effort to understand the mechanisms underlying MSTN regulation and activity with the goal of developing the most effective strategies for targeting this signaling pathway for clinical applications. Here, I review the current state of knowledge regarding the regulation of MSTN extracellularly by binding proteins and discuss the implications of these findings both with respect to the fundamental physiological role that MSTN plays in regulating tissue homeostasis and with respect to the development of therapeutic agents to combat muscle loss.

Myostatin is a TGF-β family member that normally acts to limit skeletal muscle mass. Follistatin is a myostatin-binding protein that can inhibit myostatin activity in vitro and promote muscle growth in vivo. Mice homozygous for a mutation in the Fst gene have been shown to die immediately after birth but have a reduced amount of muscle tissue, consistent with a role for follistatin in regulating myogenesis. Here, we show that Fst mutant mice exhibit haploinsufficiency, with muscles of Fst heterozygotes having significantly reduced size, a shift toward more oxidative fiber types, an impairment of muscle remodeling in response to cardiotoxin-induced injury, and a reduction in tetanic force production yet a maintenance of specific force. We show that the effect of heterozygous loss of Fst is at least partially retained in a Mstn-null background, implying that follistatin normally acts to inhibit other TGF-β family members in addition to myostatin to regulate muscle size. Finally, we present genetic evidence suggesting that activin A may be one of the ligands that is regulated by follistatin and that functions with myostatin to limit muscle mass. These findings potentially have important implications with respect to the development of therapeutics targeting this signaling pathway to preserve muscle mass and prevent muscle atrophy in a variety of inherited and acquired forms of muscle degeneration.

Myostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor β (TGFβ) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.